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A global analysis of transcription reveals two modes of Spt4/5 recruitment to archaeal RNA polymerase

Nature Microbiology volume 2, Article number: 17021 (2017) Cite this article

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Abstract

The archaeal transcription apparatus is closely related to the eukaryotic RNA polymerase (RNAP) II system, while archaeal genomes are more similar to bacteria with densely packed genes organized in operons. This makes understanding transcription in archaea vital, both in terms of molecular mechanisms and evolution. Very little is known about how archaeal cells orchestrate transcription on a systems level. We have characterized the genome-wide occupancy of the Methanocaldococcus jannaschii transcription machinery and its transcriptome. Our data reveal how the TATA and BRE promoter elements facilitate recruitment of the essential initiation factors TATA-binding protein and transcription factor B, respectively, which in turn are responsible for the loading of RNAP into the transcription units. The occupancies of RNAP and Spt4/5 strongly correlate with each other and with RNA levels. Our results show that Spt4/5 is a general elongation factor in archaea as its presence on all genes matches RNAP. Spt4/5 is recruited proximal to the transcription start site on the majority of transcription units, while on a subset of genes, including rRNA and CRISPR loci, Spt4/5 is recruited to the transcription elongation complex during early elongation within 500 base pairs of the transcription start site and akin to its bacterial homologue NusG.

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Figure 1: TSS map and promoter motif analysis.
Figure 2: Correlation between TBP/TFB binding to the promoter and RNA levels.
Figure 3: The Rpo4/7 stalk and RNAP core remain associated through the transcription cycle.
Figure 4: PIC formation and promoter strength in vitro.
Figure 5: Archaeal Spt4/5 is a general elongation factor that is recruited to RNAP via two distinct modes.
Figure 6: Initial stages of the transcription cycle in archaea.

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Acknowledgements

The authors thank J. Bähler and D. Bitton for advice throughout this project. The authors also thank T. Arnvig, D. Grohman and other members of the RNAP laboratory for encouragement and critical reading of the manuscript. Research in the RNAP laboratory at University College London is funded by Wellcome Trust Investigator Award WT096553MA (to F.W.).

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Authors and Affiliations

  1. University College London, Institute for Structural and Molecular Biology, Gower Street, London, WC1E 6BT, UK

    Katherine Smollett, Fabian Blombach & Finn Werner

  2. Institute of Microbiology and Archaea Center, Universität Regensburg, 93053 Regensburg, Germany

    Robert Reichelt & Michael Thomm

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  1. Katherine Smollett
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  2. Fabian Blombach
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Contributions

K.S. designed and performed experiments, analysed data and wrote the manuscript. F.B. performed experiments and wrote the manuscript. R.R. and M.T. helped with fermenter growth and crosslinking, and provided biomass. F.W. conceptualized the study, designed experiments and wrote the manuscript.

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Correspondence to Finn Werner.

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Supplementary information

Supplementary Information

Supplementary Notes; Supplementary Table 1 and 4–6; Supplementary Figures 1–7; Supplementary References. (PDF 8146 kb)

Supplementary Table 2

Identified TSS and their promoter elements. (XLSX 144 kb)

Supplementary Table 3

Gene organisation of Mja. (XLSX 566 kb)

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Smollett, K., Blombach, F., Reichelt, R. et al. A global analysis of transcription reveals two modes of Spt4/5 recruitment to archaeal RNA polymerase. Nat Microbiol 2, 17021 (2017). https://doi.org/10.1038/nmicrobiol.2017.21

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